The development and use of a radioimmunoassay for studies of metallothionein metabolism in animals
High-titre, avid and isoprotein-specific antibodies to the two isoproteins of rat liver Cd-metallothionein, viz. CdMT-I and CdMT-II were developed in the sheep by immunizing them with copolymers of CdMT-I or CdMT-II and rabbit IgG. A double antibody radioimmunoassay (RIA) for rat metallothionein-I (MT-I) was developed using the anti-CdMT-I antisera. The RIA did not distinguish the different metalloforms of MT-I and had a routine sensitivity of 100-200 pg/assay tube. Plasma MT-I concentrations were only 1-2 ng/ml in the adult control rats, but were considerably greater, by nearly 500-fold, in newborn rats. Up to 0.3 jig of Zn and/or Cu could therefore be bound to metallothionein/ml plasma in these young rats. Plasma MT-I concentrations were also increased in the adult rats that were injected with Cd2+, Cut+ or Zn2+, the increase being the greatest after Cd2+ injection.The degradation of rat liver metallothioneins by lysosomel extracts was studied in an in vitro system. ZnMT-I was degraded slightly more readily than ZnMT-II, but neither CuMT-I nor CuMT-II was degraded to a significant extent. This contrasts with the fast turnover rate of cytosolic CuMT in the liver of Cu-injected rats and it was shown that this apparent conflict can be partly explained by the accumulation of CuMT in the particulate fractions of the liver (probably lysosomes). Large amounts of CuMT were extracted with 2-mercaptoethanol from the particulate fractions of the livers from pigs given a Cut+-supplemented diet. Three isoproteins of CuMT were purified and fully characterized. Particulate CuMT was also isolated from the livers of rats injected with Cut+ and was shown to account for 25% of the total MT. The half-life of [35S]-CuMT in the particulate fractions (25.1 hours) was only slightly longer than that in the cytosol (20.9 hours). No CuMT was detected in the livers of rats given high -Cu2+ diets but injection of Zn caused a rapid redistribution ofthe liver Cu on to MT.